Sample analysis for mass cytometry

ABSTRACT

The invention relates to methods and devices for analysis of samples using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The invention provides methods and devices in which individual ablation plumes are distinctively captured and transferred to the ICP, followed by analysis by mass cytometry.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.14/783,791, filed on Oct. 9, 2015, which is a US National Phase of PCTApplication No. PCT/CA2014/050387, filed on Apr. 17, 2014, which claimsbenefit of U.S. Provisional application No. 61/812,893, filed Apr. 17,2013, the entire contents of which are incorporated herein by reference

TECHNICAL FIELD

This invention relates to apparatus and methods for laser ablation forcellular analysis by mass cytometry.

BACKGROUND OF THE INVENTION

Laser ablation combined with inductively coupled plasma massspectrometry (ICP-MS) can be used for imaging of biological samples(cells, tissues, etc.) labeled with elemental tags. Each laser pulsegenerates a plume of ablated material from the sample which can betransferred to be ionized for further analysis by the mass analyzer. Theinformation acquired from the laser pulses at each location on thesample can then be used for imaging the sample based on its analyzedcontent. However, this technique has limitations in its ability toseparately resolve each discrete plume of ablated material produced fromeach laser ablation pulse on the sample.

BRIEF SUMMARY OF THE INVENTION

In one aspect the invention provides a method of laser ablation masscytometry analysis comprising: directing pulses of a laser beam to asample for generating a plume of sample for each of the pulses;capturing each plumes distinctively for each of the pulses; transferringthe distinctively captured plume to an ICP; and ionizing thedistinctively captured and transferred plumes in the ICP and generatingions for mass cytometry analysis.

In a related aspect the invention provides a laser ablation masscytometer comprising: a laser ablation source for generating an ablatedplume from a sample and an injector adapted to couple the laser ablationsource with an ICP of the mass cytometer; the injector having an inletpositioned within the laser ablation source, the inlet being configuredfor capturing the ablated plume as the ablated plume is generated; and agas inlet coupled to the inlet of the injector for passing a gas therebetween for transferring the captured ablated plume into the ICP.

Also disclosed, for illustration and not limitation, are the followingexemplary aspects of the invention.

Aspect 1. A method of laser ablation mass cytometry analysis using alaser ablation mass cytometer is disclosed, the method comprising: a)directing pulses of a laser beam to a plurality of sites of a sample forgenerating an ablated plume of sample for each of the pulses; b)capturing each ablated plume distinctively; c) transferring each of thedistinctively captured ablated plumes to an inductively coupled plasma(ICP); and d) ionizing each of the distinctively captured andtransferred ablated plumes in the ICP, thereby generating ions for masscytometry analysis.

Aspect 2. The method of aspect 1, wherein the laser ablation masscytometer comprises: a laser ablation source for generating ablatedplumes from a sample; an ICP source for producing the ICP; and aninjector adapted to transfer the ablated plumes to the ICP; the injectorhaving an injector inlet positioned within the laser ablation source,the injector inlet being configured for capturing the ablated plumes;and a gas inlet coupled to the injector inlet configured to pass a gasfrom the gas inlet to the injector inlet for transferring the capturedablated plume into the ICP.

Aspect 3. The method of aspect 2 wherein the injector inlet isconfigured for capturing all or part of the ablated plume as the ablatedplume is generated.

Aspect 4. The method of any of aspects 1-3 wherein the ablated plume isgenerated by a laser pulse that is directed at a target comprising asample disposed on a substrate.

Aspect 5. The method of any of aspects 1-3 wherein the ablated plume isgenerated by a laser pulse that is directed through a transparent targetcomprising the sample.

Aspect 6. The method of aspect 5 wherein the transparent targetcomprises a transparent substrate on which the sample is situated.

Aspect 7. The method of any of aspects 2-6 wherein the injector inlethas the form of a sample cone, wherein the narrower portion of the coneis the aperture of the injector inlet.

Aspect 8. The method of aspect 7 wherein the sample cone is positionednear the area where the ablated plume is generated.

Aspect 9. The method of aspect 8 wherein the sample cone is positionedabout 100 microns away from the surface of the target surface.

Aspect 10. The method of any of aspects 7-9 wherein the diameter of theaperture a) is adjustable; b) is sized to prevent perturbation to theablated plume as it passes into the injector; and/or c) is about theequal to the cross-sectional diameter of the ablated plume.

Aspect 11. The method of aspect 7 wherein the diameter of the apertureis about 100 microns.

Aspect 12. The method of any of aspects 4-12 further comprisingintroducing a gas flow into the region between the injector inlet andthe target, to aid in directing the plume through the injector inlet.

Aspect 13. The method of aspect 13 wherein the gas flow is transverse tothe target and is transverse to the centerline of the injector lumen, atleast in the portion of the lumen proximal to the injector inlet.

Aspect 14. The method of aspect 12 or 13 wherein the target is atransparent target.

Aspect 15. The method of any of aspects 12-14 wherein the gas flowcomprises argon.

Aspect 16. The method of any of aspects 12-15 further comprisingintroducing a transfer gas flow into the injector for transferring theplume toward the ICP.

Aspect 17. The method of aspect 16 wherein the gas flow is about 0.1liters per minute and the transfer flow is about 0.9 liters per minute.

Aspect 18. The method of aspects 16 or 17 wherein the transfer flowcomprises argon.

Aspect 19. The method of any of aspects 1-4, 7-13, or 15-18 wherein thesample is on a substrate and the ablated plume is generated by a laserpulse that is directed to the sample from the same side as the sample.

Aspect 20. The method of any of aspects 2-19, wherein the gas inlet isconfigured to direct a power wash gas flow near the zone where theablated plume is formed, to direct the ablated plume towards theinjector inlet.

Aspect 21. The method of aspect 20, wherein the gas inlet comprises anozzle having an aperture smaller than the diameter of the injectorinlet.

Aspect 22. The method of any of aspects 1-21 wherein the laser beam isfrom a femtosecond laser.

Aspect 23. The method of aspect 1 wherein the ablated plume is generatedby a laser pulse that is directed through a transparent targetcomprising a transparent substrate and the sample.

Aspect 24. The method of aspect 23 wherein the laser ablation masscytometer comprises: a laser for generating ablated plumes from asample; an inductively coupled plasma (ICP) torch; an injector adaptedto transfer ablated plumes to an ICP produced by the ICP torch; whereinthe injector comprises a wall and a lumen and a portion of the injectorwall is comprised of the transparent substrate; wherein the injectorcomprises an injector inlet for introducing a gas flow into the lumenflowing, and wherein the transparent substrate is located between theinjector inlet and the ICP torch; the sample is attached to the lumenside of the transparent substrate; the ablated plumes are formed in anorientation transverse to the injector lumen and are formed entirely inthe injector lumen; and each ablated plume is distinctly captured by gasflowing through the injector lumen toward the ICP.

Aspect 25. The method of aspect 24 wherein the position of the target isfixed during analysis.

Aspect 26. The method of aspect 25 wherein directing pulses of a laserbeam to a plurality of sites of a sample comprising moving the laserbeam to sites of interest across a stationary sample.

Aspect 27. The method of aspect 26 wherein the laser beam is moved in araster pattern for imaging.

Aspect 28. The method of aspect 24 wherein the position of the target ischanged during analysis.

Aspect 29. The method of aspect 28 wherein, during analysis, the laserbeam remains stationary and the target is moved.

Aspect 30. The method of any of aspects 4-29 in which the position ofthe target is fixed during analysis.

Aspect 31. The method of aspect 30 wherein, during analysis, the laserbeam remains stationary and the target is moved.

Aspect 32. The method of any of aspects 4-29 in which the position ofthe target is moved during analysis.

Aspect 33. The method of any of aspects 1-32 wherein the laser beampulses produce 1 micron ablation spots.

Aspect 34. The method of any preceding aspect wherein thecross-sectional diameter of the ablated plume is on the scale of 100microns.

Aspect 35. The method of any preceding aspect wherein the injector is atube with an approximately 1 mm inner diameter.

Aspect 36. The method of any preceding aspect wherein the ablated plumesformed by each laser pulse comprise sample particles with dimensions ofabout 1 μm or less.

Aspect 37. The method of any of preceding aspect wherein spreading ofthe ablation plume as it is transferred to the ICP is maintained withinthe internal diameter of the injector lumen.

Aspect 38. A laser ablation mass cytometer comprising: a laser ablationsource for generating ablated plumes from a sample; a laser that emits alaser beam from a surface, the surface oriented to direct the beam to asample contained in the laser ablation source; an inductively coupledplasma (ICP) torch; an injector adapted to couple the laser ablationsource with an ICP produced by the ICP torch; the injector having aninjector inlet positioned within the laser ablation source, the injectorinlet being configured for capturing the ablated plume as the ablatedplume is generated; and a gas inlet coupled to the injector inlet of theinjector inlet configured to pass a gas from the gas inlet to theinjector inlet for transferring the captured ablated plume into the ICP.

Aspect 39. The cytometer of aspect 38 configured so that the laser beamis oriented directly toward the opening of the injector inlet.

Aspect 40. The cytometer of aspect 39 configured so that the laser beamis aligned with the lumen of the injector at least at the portion of thelumen proximal to the injector inlet.

Aspect 41. The cytometer of aspect 39 configured so that a projection ofthe laser beam is transverse to the centerline of the injector lumen, atleast in the portion of the lumen proximal to the injector inlet.

Aspect 42. The cytometer of any of aspects 38-41 wherein the laserablation source is adapted to receive a transparent target.

Aspect 43. The cytometer of aspect 42 further comprising a transparenttarget.

Aspect 44. The cytometer of aspects 42 or 43 wherein the transparenttarget comprises a transparent substrate and the sample.

Aspect 45. The cytometer of any of aspects 38-44 wherein the diameter ofthe aperture of the injector inlet is less than the inner diameter ofthe injector.

Aspect 46. The cytometer of any of aspects 38-44 wherein the injectorinlet has the form of a sample cone.

Aspect 47. The cytometer of aspect 46 wherein the sample cone ispositioned near the zone where ablation plumes are generated.

Aspect 48. The cytometer of aspect 46 wherein the diameter of theaperture is adjustable.

Aspect 49. The cytometer of any of aspects 45-48 comprising atransparent target.

Aspect 50. The cytometer of any of aspects 38-49 further comprising agas flow inlet configured to direct gas in an orientation transverse tothe centerline of the lumen of the injector at least at the portion ofthe lumen proximal to the injector inlet.

Aspect 51. The cytometer of any of aspects 38-50 further comprising agas flow inlet configured to direct gas across the surface of thetransparent target toward the aperture, to aid in directing an ablationplume through the injector inlet.

Aspect 52. The cytometer of aspect 51, wherein the injector inlet hasthe form of a sample cone, further comprising a transfer gas flow inletpositioned configured to direct gas into the lumen of the injector.

Aspect 53. The cytometer of aspect 38 comprising a power wash gas inletconfigured to direct ablated plumes into the injector inlet.

Aspect 54. The cytometer of aspect 53 wherein the power wash gas inletcomprises a nozzle having an aperture smaller than the aperture of theinjector inlet.

Aspect 55. A laser ablation mass cytometer comprising: a femtosecondlaser for generating ablated plumes from a sample; an inductivelycoupled plasma (ICP) torch; an injector adapted to transfer ablatedplumes to an ICP produced by the ICP torch; wherein the injectorcomprises a wall and a lumen, and a portion of the injector wall iscomprised of the transparent substrate, said transparent substrateadapted to receive the sample; wherein the injector comprises aninjector inlet for introducing gas into the lumen, wherein thetransparent substrate is located between the injector inlet and the ICPtorch.

Aspect 56. The cytometer of aspect 55 wherein the transparent substrateis movable relative to other portions of the injector wall.

Aspect 57. The cytometer of aspect 56 wherein the transparent substratecan be moved in a raster pattern relative to other portions of theinjector wall.

Aspect 58. A laser ablation system comprising a) a laser capable ofproducing laser illumination; b) a laser ablation cell comprising atransparent substrate for holding a sample to be analyzed or a stageconfigured to receive a transparent substrate; and c) an injector forcarrying an ablation plume to an ICP, said injector comprising aninjector opening, wherein the (a), (b) and (c) are configured so thatthe laser illumination originates on one side of the stage or substrateand the injector opening is on the other side.

Aspect 59. The system of aspect 58 in which the laser illuminationpassed through an optical window into the ablation cell.

Aspect 60. The system of aspect 59 in which the injector opening isconfigured so that the ablation of an area of the substrate results inan ablated plume formed downstream of a surface from which the laserillumination is emitted.

Aspect 61. The system of aspect 60 in which the surface is a lens ormirror.

Aspect 62. The system of aspect 61 in which the injector opening isconfigured so that the ablation of an area of the substrate results inan ablated plume formed at least partially in the injector.

Aspect 63. The system of any of aspects 58-62 comprising (a) a transfergas source for producing a transfer flow in the injector, (b) a capturegas source for producing a capture flow in the ablation cell, or both(a) and (b).

Aspect 64. The system of any of aspects 58-63 wherein the stage moves inx-y or x-y-z directions.

Aspect 65. The system of any of aspects 58-63 comprising a biologicalsample on the transparent substrate.

Aspect 66. The method of any of aspects 7-11 wherein the laser beampasses through said aperture.

Aspect 67. The method of aspect 66 in which the ablation plume expandstowards the surface from which the laser beam emanates.

Aspect 68. A laser ablation inductively coupled plasma mass spectrometrysystem comprising: a laser ablation source for generating an ablatedplume from a sample; a laser that emits a laser beam, wherein said beampasses through an objective lens to a sample contained in the laserablation source; an inductively coupled plasma (ICP) torch; and, aninjector adapted to couple the laser ablation source with an ICPproduced by the ICP torch; wherein the injector passes though an openingin the objective lens; the injector having an injector inlet positionedwithin the laser ablation source, the injector inlet being configuredfor capturing the ablated plume as the ablated plume is generated.

Aspect 69. The system of aspect 68 wherein the laser beam is reflectedfrom a mirror to the objective lens.

Aspect 70. The system of aspect 69 wherein the injector passes throughan opening in the mirror.

Aspect 71. The system of any of aspects 68-70 wherein the ablationsource comprises an inlet for a capture gas flow.

Aspect 72. The system of any of aspects 68-71 wherein the ablationsource comprises a stage configured to receive a target.

Aspect 73. A laser ablation inductively coupled plasma mass spectrometrysystem configured for use according to any method disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled person in the art will understand that the drawings,described below, are for illustration purposes only. The drawings arenot intended to limit the scope of the applicant's teachings in any way.

FIG. 1 is a schematic view of a laser ablation mass cytometer.

FIG. 2 is a diagrammatic view of an embodiment of the laser ablationsource of FIG. 1 showing the sampling of the laser ablated plume throughan aperture configured for transferring the plume into an injector.

FIG. 3 is a view of an alternative configuration similar to FIG. 2 withthe plume sampled directly into the injector.

FIG. 4 and FIG. 5 are diagrammatic views of further various embodimentsof the laser ablation source of FIG. 1 showing the generation and thesampling of the laser ablated plume within the injector.

FIG. 6 is a view of an alternative configuration similar to FIG. 2 butshowing a ‘power wash’ flow directed normal to the plume formation todirect the plume for transfer into the injector.

FIG. 7 shows an embodiment where the sample under study is illuminatedby the laser light from the top side.

FIG. 8 shows an embodiment in which a part of the sheath flow isdiscarded as a sacrificial flow while the core of the sheath flowcontaining capture flow and plume material enters.

FIG. 9 shows an arrangement in which the plume is sampled into aninjector that passes through the objective lens.

FIG. 10 shows an arrangement in which the plume is sampled into aninjector that passes through the objective lens and a mirror.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

It should be understood that the phrase “a” or “an” used in conjunctionwith the present teachings with reference to various elementsencompasses “one or more” or “at least one” unless the context clearlyindicates otherwise.

The present invention relates to laser ablation combined withinductively coupled plasma mass spectrometry (LA-ICP-MS). LA-ICP-MS hasbeen described for measurement of endogenous elements in biologicalmaterials and, more recently, for imaging by detection ofelemental-tagged antibodies. See, e.g., Antonov, A. and Bandura, D.,2012, U.S. Pat. Pub. 2012/0061561, incorporated by reference herein;Seuma et al., “Combination of immunohistochemistry and laser ablationICP mass spectrometry for imaging of cancer biomarkers” 2008, Proteomics8:3775-3784; Hutchinson et al. “Imaging and spatial distribution ofβ-amyloid peptide and metal ions in Alzheimer's plaques by laserablation—inductively coupled plasma-mass spectrometry” Analyticalbiochemistry 2005, 346.2:225-233; Becker et al. “Laser ablationinductively coupled plasma mass spectrometry (LA-ICP-MS) in elementalimaging of biological tissues and in proteomics.” 2007, Journal ofAnalytical Atomic Spectrometry 22.7:736-744; Binet, et al., “Detectionand characterization of zinc- and cadmium-binding proteins inEscherichia coli by gel electrophoresis and laser ablation-inductivelycoupled plasma-mass spectrometry” Analytical Biochemistry 2003,318:30-38; Quinn, et al., “Simultaneous determination of proteins usingan element-tagged immunoassay coupled with ICP-MS detection Journal ofAnalytical Atomic Spectrometry” 2002, 17:892-96; Sharma, et al.,“Sesbania drummondii cell cultures: ICP-MS determination of theaccumulation of Pb and Cu Microchemical Journal” 2005, 81:163-69; andGiesen et al. “Multiplexed immunohistochemical detection of tumormarkers in breast cancer tissue using laser ablation inductively coupledplasma mass spectrometry” 2011, Anal. Chem. 83:8177-8183, each of whichis incorporated by reference herein.

The present invention provides methods of laser ablation mass cytometryanalysis in which pulses of a laser beam are directed to a sample forgenerating a plume of sample for each of the pulses; capturing eachplume distinctively for each of the pulses; transferring each of thedistinctively captured plume to an ICP; and ionizing each of thedistinctively captured and transferred plumes in the ICP and generatingions for mass cytometry analysis and devices for carrying out themethod. In various embodiments, a laser ablation mass cytometer can havea laser ablation source for generating an ablated plume from a sampleand an injector adapted to couple the laser ablation source with an ICPof the mass cytometer. In some embodiments the injector can have aninlet positioned within the laser ablation source such that the inletcan be configured for capturing the ablated plume as the ablated plumeis generated. A gas inlet can be coupled to the inlet of the injectorfor passing a gas there between for transferring the captured ablatedplume into the ICP.

In one aspect the invention provides a laser ablation mass cytometerthat has (i) a laser ablation source (ii) an injector adapted to couplethe laser ablation source with an ICP produced by an ICP source; and(iii) a mass analyzer.

The laser ablation source, also referred to as the “ablation cell,”houses the sample during ablation. Typically the ablation cell includesa laser transparent window to allow laser energy to strike the sample.Optionally the ablation cell includes a stage to hold the sample to beanalyzed. In some embodiments the stage is movable x-y or x-y-zdimensions. In drawings and examples herein, the laser ablation sourceis sometimes shown as an open arrangement. However, such configurationsare for illustration only, and it will be recognized that some form ofsuitable enclosure for preventing contamination or infiltration from theambient environment is present. For example, a chamber configured withgas inlets and/or optical ports can be arranged around the laserablation source to provide an enclosed environment suitable forcapturing and transferring the ablated plume for ICP mass analysis. Thegas inlets and optical port(s) are positioned so that the orientation ofthe laser beam, sample, plume expansion, and injector are suitable forthe methods and devices disclosed herein. It will be appreciated thatthe ablation cell is generally gas tight (except for designed exits andports).

Lasers used for laser ablation according to the invention generally fallinto three categories: femtosecond pulsed lasers, deep UV pulsed lasersand pulsed lasers with a wavelength chosen for high absorption in theablated material (“wavelength selective lasers”). Deep UV and wavelengthspecific lasers would likely operate with nanosecond or picosecondpulses. Each class of lasers has its drawbacks and benefits and can bechosen based on a particular application. In some embodiments, the laseris a femtosecond pulsed laser configured to operate with a pulse ratebetween 10 and 10000 Hz. Femtosecond laser are known (see, e.g., Jhaniset al., “Rapid bulk analysis using femtosecond laser ablationinductively coupled plasma time-of-flight mass spectrometry” J. Anal.At. Spectrom., 2012, 27:1405-1412.

Femtosecond lasers allow for laser ablation of virtually all materialswith the only prerequisite for laser ablation being-sufficient powerdensity. This can be achieved even with relatively low pulse energy whenthe beam is tightly focused, for instance to 1 micrometer diameter andis short in duration (focused in time). Deep UV lasers also can ablate alarge class of materials because most of the commonly used materialsabsorb deep UV photons. Wavelength selective laser ablation can utilizethe lasers with the specific laser wavelength targeting absorption inthe substrate material. A benefit of the wavelength specific laser maybe the cost and simplicity of the laser and the optical system, albeitwith a more limited spectrum of substrate materials. Suitable lasers canhave different operating principles such as, for example, solid state(for instance a Nd:YAG laser), excimer lasers, fiber lasers, and OPOlasers.

A useful property of the femtosecond laser light is that it is absorbedonly where the threshold power density is reached. Thus, a convergingfemtosecond laser light can pass through a thicker section of materialwithout being absorbed or causing any damage and yet ablate the samematerial right at the surface where the focus is occurring. The focuscan then be moved inside the material progressively as the sample layersare ablated. Nanosecond laser pulses might be partially absorbed by thesubstrate but can still work for ablation since the energy density atthe focal point will be the highest (as long as it is sufficient forablation).

The laser pulse may be shaped using an aperture, homogenized (ifrequired) using a beam homogenizer, focused, e.g., using an objectivelens, to produce a desired spot size less than 10 μm. Exemplary spotsizes include diameters (or equivalent sized ablation areas of othershapes) in the range of 0.10-3 μm (e.g., about 0.3 μm), 1-5 μm (e.g.,about 3 μm), 1-10 μm (e.g., about 1, about 2, about 3, about 4 or about5 μm), less than 10 μm, and less than 5 μm. In particular embodiments, alaser system is configured to operate with sufficiently focused laserpulses to ablate a sample area in the order of about 1 μm, e.g., 100 nmto 1 μm. Ablation on this small scale produces very small amount ofplume material that in turn ensures that the size of the plume is keptsmall. A smaller plume is more likely to stay in the middle of thecapture flow without contacting the walls of the ablation cell or of theinjector gas conduits. Ablation on the 1 micrometer scale also meansthat the distance between the ablated surface and the area where plumeexpansion slows down and becomes dominated by the ambient gas is veryshort. This distance can range from a few micrometers to a few hundredmicrometers. In some versions of the invention, the capture flow ispresent where the plume stops expanding. Therefore, for illustration andnot limitation, several of the appended figures show the distancebetween the ablated surface and the region with capture flow shown asabout 100 micrometers.

Although ablation on the 1 micrometer (or lower) scale is advantageousfor certain applications (e.g., imaging), the methods and instruments ofthe invention are also useful when larger ablation spots are produced,such as ablation spots in the range of about 5 to about 35 micronsdiameter, for example in the range 5-15 microns, 10-20 microns, 15-25microns, 20-30 microns and 25-35 microns. In some applications in whichlarge ablation spots are produced, only a portion of the plume materialis captured.

In some embodiments, the laser is situated outside the laser ablationsource, and the laser beam (laser energy) enters the laser ablationsource, e.g., though an optical window. As used herein, a laser beam maybe describes as being emitted from a surface (e.g., a laser lens ormirror), which surface may be oriented to direct the beam to aparticular location or pattern of locations. For ease of description ofthe invention, the directed beam may be considered to have a particularorientation; the orientation of the beam can refer to an imaginary linealigned with the beam and extending beyond the actual beam (for examplewhen the beam strikes a non-transparent surface). As will be apparentfrom context, reference to the orientation or position of a laser beamsometimes refers to the orientation or position the beam of an unpoweredlaser source would produce if the laser was in use.

Mass analyzers for use in the invention may be selected based on theneeds of the operator or specific application. Exemplary types of massanalyzers include quadrupole, time of flight, magnetic sector, highresolution, single or multicollector based ICP mass spectrometers.Typically, time of flight mass spectrometers are used for the recordingof fast transient events with the transit durations that are expectedfrom the fast laser ablation ICP setup.

Ions are produced when particles of the ablation plume enter plasma(inductively coupled plasma, ICP) maintained within an ICP source or ICPtorch.

A mass cytometer may be used for analysis or imaging of a biologicalsample, which may be on transparent substrate. In imaging embodiments,generally the laser may be operated with continuous train of pulses orin bursts of pulses directed to different positions of the sample,referred to as “spots of interest,” or “locations or zones of ablation.”The pulses may be directed to spots in a set pattern, such as a rasterfor two-dimensional imaging. Alternatively, a plurality of individualspots at different locations (for example, corresponding to individualcells) may be ablated. In some embodiments, the laser emits a burst ofpulses producing a plume coming from the same pixel (i.e. the samelocation on the target). Ablation plumes produced by individual pulseswithin the burst are expected to fuse into one plume and travel withinthe instrument in such a way that they will be distinct from the plumeproduced from another pixel. To distinguish individual pixels, the timeduration between bursts (pixel interrogation that can be just one pulseor 100 pulses) is maintained above a certain limit determined by thetime spreading of the ion signal (at the detector) from an individualpixel.

As described below, one feature of the invention is that the ablationplume is transferred from the site of plume formation to the ICP in aprocess that allows each separate sample plume to be distinctlyanalyzed. The plume is transported from the zone of formation to the ICPthrough, at least in part, a conduit or injector tube (“injector”). Thetube may be formed, for example, by drilling through a suitable materialto produce a lumen (e.g., a lumen with a circular, rectangular or othercross-section) for transit of the plume. An injector tube sometimes hasan inner diameter in the range 0.2 mm to 3 mm. In some embodiments theinjector conduit has a smaller diameter, for example when incorporatedwith or into a microfluidic device. In some embodiments, the innerdiameter of the injector varies along the length of the injector. Forexample, the injector may be tapered at an end. An injector sometimeshas a length in the range of 1 centimeter to 100 centimeters. In someembodiments the length is no more than 10 centimeters (e.g., 1-10centimeters), no more than 5 centimeters (e.g., 1-5 centimeters), or nomore than 3 cm (e.g., 0.1-3 centimeters). The injector may be formed,without limitation, from metal (e.g., steel), quartz, glass, sapphire orother materials. In some embodiments the injector lumen is straightalong the entire distance, or nearly the entire distance, from theablation source to the ICP. In some embodiments the injector lumen isnot straight for the entire distance and changes orientation. Forexample, the conduit may make a gradual 90 degree turn. Thisconfiguration allows for the plume to move in a vertical plane initiallywhile the axis at injector inlet will be pointing straight up, and movehorizontally as it approached the ICP torch (which is commonly orientedhorizontally to take advantage of convectional cooling). In someembodiments the injector is straight for a distance of least 0.1centimeters, at least 0.5 centimeters or at least 1 centimeter from theaperture though which the plume enters or is formed.

As used herein, the “centerline” of an injector lumen is an imaginaryline through the center of, and extending out of, the lumen, optionallya line following an axis of symmetry, and is a useful reference fororientation. For example, a laser beam, the orientation of plumeexpansion, and centerline may be aligned with each other. In anotherexample, the orientation of plume expansion may be transverse (e.g.,orthogonal) relative to the centerline.

In accordance with the present teachings, each separate sample plume canbe distinctly analyzed by the mass analyzer. In one aspect, the deviceis configured so that spreading of the plume in ablation cell (ablationsource) and injector is smaller than the spreading that occurs in theICP source and the mass analyzer. In one aspect, plumes may bedistinctly analyzed by transferring each ablated plume to the ICP in atime period that is within the cumulative transit time of the plume tothe ICP and ion detection by the mass analyzer. This can be accomplishedby capturing each sample plume through a gas flow and under a transferconfiguration such that the ratio between the plume broadening duringtransfer time period (i.e., transfer of the ablation plume from the siteof ablation to the plasma) and the broadening during ion transit timeperiod (i.e., transfer of ions from the plasma to the mass analyzer) isequal to or less than one.

Generally, the sample particle size limit for which an ICP ion sourcecan effectively vaporize and ionize for the purpose of analyticaldetection is in the order of about 10 μm or less. Particles produced bythe laser ablation at 1 micrometer scale are below 1 micrometer and arewell suited for ICP ion source. For discrete particles analysis (such asmay be carried out using CyTOF® instrumentation, Fluidigm Canada Inc.),the typical rate at which these particles can be ionized andanalytically detected can be a function of the cumulative broadening orspread of transit time of the sample in the plasma while the particlesare being evaporated and ionized and of the ions' transit timebroadening or spread between the ICP and its detection by the massanalyzer. Generally the cumulative time broadening or spread can be ofthe order of about 200 μs duration. Consequently, for particles of 10 μmor less that are spatially separated, analyzing each distinct particlecan be achieved by transferring each particle to the ICP in a timeperiod of the order of 200 μs. In some embodiments the particles aretransferred to the ICP in less than 200 μs, or less than 150 μs.Accordingly, in a sample introduction system where imaging of biologicalsamples can be performed by laser ablation, a laser system can beconfigured to operate with sufficiently focused laser pulses to ablate asample area in the order of about 1 μm, such as the application of afemtosecond pulsed laser for example. With this configuration, theablated plumes formed by each laser pulse can include sampleparticulates with dimensions typically about 1 μm or less. Under certainconditions as described herein, these particulates can be captured andtransferred to meet the transfer time period as required and,subsequently, each distinct plume can be effectively vaporized andionized by the ICP.

Additionally, while operating the laser with continuous series of pulsessuch as in the case of rasterizing across a sample surface for twodimensional imaging, the distinctiveness of each plume and the spatialseparation between each subsequent plume can be maintained between theplume's zone of formation and the point of vaporization and ionizationin the ICP ion source. For example, as a plume is carried through aconduit, such as the injector tube shown in FIG. 1, the particles in theplume can spread and expand outwardly in a radial direction before itenters the plasma of the ICP. Spreading of the particles produced in theplume can depend on its diffusion coefficient, the velocity profile ofcarrier flow and the distribution of particle density as it is formedand as it evolves during transit to the ICP. For example, thefemtosecond laser ablation spot size of 1 μm can produce a plume with aninitial cross section diameter of about 100 μm or less before furtherspreading during its transit. The extent of spreading of the plume canalso be a function of the dimension of the ablated particle; largerparticles tend to have lower diffusion spreading but with highermomentum resulting in potential losses due to contacting the inner wallsof the injector tube. It is thus desirous to minimize the plumespreading and/or to transfer the plume to the ICP within sufficient timeto vaporize and ionize before the extent of spreading presents anychallenging effects.

Accordingly, in various embodiments, the use of a laser for ablating 1μm sample spots and efficiently transporting the plume so that thespreading is maintained within the internal diameter of the injectortube can be achieved by the exemplary arrangements described herein andin the accompanying drawings.

For a given laser ablation system and given sample, ablated plumesexpand after the laser ablation until they reach a characteristicvolume, referred to as the “sampling volume.” It is desirable toconfigure the system to minimize the sampling volume, and to increasethe velocity with which the gas flow carries the plume away from thesampling volume. The combination of a small sampling volume and fast gasflow reduces the time spreading of the plume transfer into the injector.The sampling volume can be described by the envelope of the plume at themoment when the velocity of plume expansion in any of the dimensionsfalls substantially (˜10 times) below the sonic velocity of thesurrounding gas media. Without limitation, exemplary sampling volumesmay be in the rang 10⁻⁶ mm³-10 mm³. Often the sampling volume is in therange 0.001 mm³-1 mm³. The capture flow, where present, flows into atleast part of the sampling volume and carries at least a portion of theplume into the injector whereupon it may be transported by the transferflow to the IPC. It is desirable that the velocity of capture flow whenit enters the sampling volume be substantial (e.g., >1 m/s, >10m/s, >100 m/s, or >500 m/s). In some embodiments the velocity of captureflow when it enters the sampling volume can be estimated by measuringthe velocity of the capture flow into the injector (e.g., though theinjector aperture). In some embodiments this measured velocity is >1m/s, >10 m/s, >100 m/s, or >500 m/s. In contrast to the presentinvention, if the plume is not swept away rapidly, it will continue toexpand and diffuse, undesirably filling the entire ablation cell.

In one aspect In one aspect, the invention provides a laser ablationconfiguration in which the laser beam is directed to a target. In oneembodiment, the target comprises a substrate and a sample disposed onthe substrate. In one embodiment the substrate is transparent and thetarget is a transparent target.

In one aspect, the invention provides a laser ablation configuration(discussed below in the context of, but not limited to, FIG. 2), for“through-target” ablation. In this configuration, the pulse of a laserbeam is directed through the transparent target and a sample plume (the“ablated plume” or the “plume”) is formed downstream of the beam into aninjector. Also see FIGS. 3-5. Through-target illumination isadvantageous for optimizing transit time broadening due to the removalof optical elements (windows, objective lenses, etc.) from the straightpath of the plume. In one aspect, the invention provides a laserablation system comprising (a) a laser capable of producing laserillumination; (b) a laser ablation cell (or laser ablation source) intowhich a transparent target may be introduced and an injector with anopening through which an ablated plume may enter, where the laserillumination originates from a surface on one side of the transparenttarget and the injector opening is on the other side. Other featuresthat may be included in the system are described throughout thisdisclosure including the examples.

In FIG. 1, a laser ablation mass cytometer comprises a laser ablationsource that can be connected to an injector, such as a tube fabricatedfrom quartz or other generally suitable material, and mounted for sampledelivery into an inductively coupled plasma (ICP) source, also referredto as an ICP torch. The plasma of the ICP torch can vaporize and ionizethe sample to form ions that can be received by a mass analyzer.

In various embodiments according to FIG. 2, the sample of interest canbe configured for laser ablation by using a sample formatted to becompatible with a transparent target. A sample can be placed onto atransparent substrate, incorporated into a transparent substrate or canbe formed as the transparent target. Suitable laser-transparentsubstrates may comprise glass, plastic, quartz and other materials.Generally the substrate is substantially planar or flat. In someembodiments the substrate is curved. Substrates are from 0.1 mm up to 3mm thick, in certain embodiments. In some embodiments, the substrate isencoded (see, e.g., Antonov, A. and Bandura, D., 2012, U.S. Pat. Pub.2012/0061561, incorporated by reference herein). In this configuration,the pulse of a laser beam is directed through the transparent target anda sample plume (the “ablated plume” or the “plume”) is formed downstreamof the beam into an injector.

The injector, or injector tube, can have an inlet configured to capturethe ablated plume; such as the inlet formed as a sample cone having asmall opening or aperture as illustrated in FIG. 2. In thisconfiguration, the sample cone can be positioned near the area, or zone,where the plume is formed. For example, the opening of the sample conemay be positioned from 10 μm to 1000 μm from the transparent target,such as about 100 μm away from the transparent target. Consequently, theablated plume can be generated and formed at least partially within theexpanding region of the cone. In some embodiments, the diameter of theaperture and/or dimensions of the spacing (including angles) areadjustable to permit optimization under various conditions. For example,with a plume having a cross sectional diameter in the scale of 100 μm,the diameter of the aperture can be sized in the order of 100 μm withsufficient clearance to prevent perturbation to the plume as it passes.

The injector can continue downstream of the sampling cone for receivingthe ablated plume in such a configuration as to encourage the movementof the plume and preserve the spatial distinctiveness of each subsequentplume as a function of the laser pulses. Accordingly, a flow of gas canbe introduced to aid in directing the plume through the aperture of thesampling cone in order to capture (capture flow) each plumedistinctively while an additional flow of gas can be introduced to theinjector for transferring (transfer flow or sheath flow) each distinctlycaptured plume towards the ICP. Another function of the transfer orsheath flow is to prevent the particles produced in the plume fromcontacting the walls of the injector. The gas(es) may be, for example,and without limitation, argon, xenon, helium, nitrogen, or mixtures ofthese. In some embodiments the gas is argon. The capture flow gas andthe transfer flow gas may be the same or different.

It is within the ability of one of ordinary skill in this field guidedby this disclosure to select or determine gas flow rates suitable forthe present invention. The total flow through the injector is typicallydictated by the requirements of the ICP ionization source. The laserablation setup needs to provide the flow that would match theserequirements. For example, in FIG. 2, as well as other figuresillustrating various configurations, the injector tube has beengenerally described with a 1 mm inner diameter in conjunction with thecumulative gas flow rate of about 1 liter per minute (0.1 liter perminute capture flow plus 0.9 liter per minute transfer flow). It wouldbe expected that smaller or larger diameter injectors, along with thecorrespondingly selected gas flow rates, can be applied to the variousgeometries presented with similar expected results. Conditions formaintaining non-turbulent gas dynamic within the injector tube in orderfor preserving the distinctiveness of each separate ablated plume aredesirable.

As described herein, given a particular configuration of elements (e.g.,a particular configuration of gas inlet positions, apertures, injectorproperties, and other elements), the capture and transfer flow rates areselected to result in transfer of each ablated plume to the ICP in atime period that is within the cumulative transit time of the plumebetween the ICP and its detection by the mass analyzer. This can beaccomplished by capturing each sample plume through a gas flow and undera transfer configuration such that the ratio between the plumebroadening during transfer time period and the broadening during iontransit time period is equal to or less than one. That is, the timebroadening (or time spreading) of the transit signal that is important.ICP-MS devices (such as the CyTOF® ICP-TOF instrument, Fluidigm CanadaInc.) are characterized by an inherent broadening of the signal. In thecase of laser ablation, the act of injecting a single plume may or maynot be fast in comparison to the time spreading on the ICP-MS itself.The spreading of the plume before plasma depends on the design of theablation cell and plume delivery channel (injector). It is desirablethat the laser ablation cell and its sample delivery system (injector)does not spread the original ablation plume more than the inherentbroadening of the remaining instrument. This condition ensures that thespike in detection signal produced by ablation plume is as sharp (intime) as it could be for the chosen instrument. If the spreading of theplume is much longer then the spreading in the ICP-MS, an event of laserablation from a single pulse will come out much broader at the detector.But, if the spreading in the laser ablation section is smaller than theinstrument spreading the total spreading will be dominated by theinstrument spreading. Thus, one can measure the instrument spreadingusing calibration beads and then measure the total spreading from asingle laser pulse and compare these two numbers. If the spreading fromthe laser ablation is smaller than the spreading from the instrument,the total spreading will be less than 2-times of the instrumentspreading.

The characteristic instrument time broadening can be measuredexperimentally, for example using labeled cells or calibration beads.Any time a single bead enters a mass cytometer (e.g., CyTOF® ICP-TOFinstrument) the bead goes through evaporation and ionization in plasmaand then goes through the mass analyzer until its signal reachesdetector. The transient event is detected and used to record informationabout the particular bead, such as the width of the transient signal(which represents the time spread from a single event) and the value ofspreading that occurs starting from the ICP source and ending at thedetector.

In some embodiments, the device is configured to allow time spreading ofbetween 10 and 1000 microseconds for the path defined between the sampleand the ion detector of the mass analyzer.

Typical capture flow rates are in the range of 0.1 to 1 Lpm. An optimalcapture flow rate can be determined experimentally, but is usually atthe lower end of the range (e.g., about 0.1 Lpm). Typical transfer flowrates are in the range of 0.1 to 1 Lpm. An optimal transfer flow ratecan be determined experimentally, but is usually at the higher end ofthe range (e.g., about 0.9 Lpm). In some embodiments, the capture flowrate is lower than the transfer flow rate. The transfer flow rate can be0 in some cases, for example if the capture flow rate is approximately 1Lpm. Often the transfer flow rate is in the range of 0.4-1 Lpm (e.g.,0.4, 0.6, 0.8 or 1 Lpm).

For illustration, in the configuration shown in FIG. 2, the flow rate ofthe gas supplied for capturing the plume through the sampling cone canbe about 0.1 liters per minute while the transfer flow of about 0.9liters per minute can pass through a 1 mm inner diameter injector tube.The gas flows and their introduction orientation can be optimized foreffective capture and transfer of each ablated plume so that each plumemaintains its distinctiveness.

In various embodiments according to FIG. 3, the sampling cone of FIG. 2can be omitted so that an open ended injector can be positioned in placeof the aperture. In this configuration the accumulative flow rate ofabout 1 liter per minute of the supply gas can be introduced in such away as to be able to capture and to transfer each ablated plumedistinctly and directly into the injector. In some embodiments thedistance between the surface of the transparent target and the injectorinlet is 500 μm or less, such as less than about 200 μm, less than about100 μm or less than about 50 μm. In the configuration of FIG. 3, thereis no separate capture flow and transfer flow. Instead, a single gasflow directs the plume through the aperture and transfers the distinctlycaptured plume towards the ICP. In this arrangement, the gas flow isoften in the range of 0.2 liters per minute to 2 liters per minute.

In various embodiments, the ablated plume can be formed directly withinthe injector tube with its direction of formation oriented in thetransverse direction as indicated in FIG. 4 and FIG. 5. With the similartransparent target configuration as described according to FIG. 2, eachablated plume can be captured by the gas flow (about 1 liter per minute)and drawn downstream to the ICP. Since the transparent targetillustrated in FIG. 4 is in a fixed position with respect to theinjector tube, the location of each ablation spot can be varied toprovide scanning capabilities. For example, the incident laser beamablation can be moved to various spots of interest across the stationarysample or moved in a raster pattern to provide greater imagingcapability. Generally in raster operation, the pulsed laser operatescontinuously as the location of ablation changes according to a setpattern. Alternatively, in various embodiments, the laser beam canremain stationary while the target can be configured for movement toprovide different spots for the ablation as illustrated in FIG. 5.

In various embodiments according to FIG. 6, the laser beam can bedirected incident onto the target from the same side as the sample. Inthis instance, the sample can be placed on a substrate and each pulse ofthe laser beam can generate the ablated plume expanding in the directionof the incident laser. The laser light might be about orthogonal to thesubstrate or may be oriented at other angles, which will result inablation spot that is stretched (for instance, elliptical instead ofround). A constrain to the laser light angle is that the light itselfconverges in a cone. Focusing of the beam to 1 micrometer scale requiresthe cone angle to be quite wide (often expressed as operating at highnumerical aperture). This means that significant tilting of the laserbeam might affect the ability to focus the laser to a tight spot.

FIG. 6 illustrates the use of a “power wash.” A ‘power wash’ flow of gascan be directed near (e.g., at about 100 μm distance away) the zone fromwhich the plume is formed. The gas flow from the ‘power wash’ can forcethe ablated plume, or redirect the plume, towards the inlet end of theinjector tube, effectively capturing each plume as it is formed orgenerated. With the similar configuration as described according to theabove examples, the injector tube can be provided with a gas flow (about0.9 liters per minute in this illustration) to capture and transfer theplume towards the ICP. In various embodiments for example, the ‘powerwash’ flow can be achieved with a flow of gas (about 0.1 liter perminute) delivered. through a narrow nozzle (about 100 μm in diameter forexample) for creating a gas jet suitable for redirecting each subsequentablated plume into the injector tube. The source of the power wash gasflow (e.g., nozzle) can be referred to as a “gas inlet,” because it isan inlet of the power wash gas flow toward the plume. Alternatively thesource of the power wash gas flow can be referred to as a “port.” Forexample, the ‘power wash’ flow of gas can emerge from a nozzle at adistance of 50 μm to 200 μm from the laser ablation spot (the zone offormation of the plume). It will be clear that, as used in this context,“nozzle” does not refer to any particular structure, but refers to theoutlet from which the power wash gas emerges. As illustrated in FIG. 6,the diameter of the power wash nozzle is smaller than the inner diameter(or equivalent cross-sectional dimension) of the injector. For example,the diameter of the nozzle may be from 10% to 50% of the diameter of theinjector. In some embodiments the power wash directs the plume into acone-shaped injector inlet.

FIG. 7 shows an embodiment where the sample under study is illuminatedby the laser light from the top side. The laser light is focused by anobjective then passes through an optical window and finally enterssealed ablation chamber through a conical conduit. The conical shape ofthe conduit allows for the laser light to pass to the target whileproviding a conduit for the capture gas to exit the chamber. The capturegas carries the content of ablation plume and then merges with thesheath flow. By choosing dimensions of the gas channels and flow ratesone can ensure that the capture flow gets surrounded by the sheath flowand that the plug from an ablation plume stays near the axis of theinjector flow. This location of the plume facilitates the fastesttransfer of the plume with reduced time spreading.

FIG. 8 shows a configuration similar to that of FIG. 7 and illustratesthat a stronger sheath flow may be used to surround flow with plumematerial in the center of the flow. FIG. 8 illustrates that a part ofthe sheath flow is discarded as a sacrificial flow while the core of thesheath flow containing capture flow and plume material enters a shortconduit that supplies this flow into the ICP.

The technique of utilizing sacrificial flow illustrated in FIG. 8 can beapplied to other configurations described above. In such embodiments theinjector can be considered to have two portions with different innerdiameters. A major benefit of sacrificial flow configuration is that thecapture flow and the plume material stay near the center of the tubingwhere velocity profile of the gas flow is nearly flat, i.e. differentparts of the captured plume advance with similar velocities.

FIG. 9 shows another embodiment with laser beam illumination on top ofthe sample. Here the plume is sampled into the sampling conduit arrangedabout normal to the target. The plume material is surrounded by thecapture flow that also acts as a sheath flow. The gas dynamics of thecapture of the plume in FIG. 9 resembles that of FIG. 3 wherethrough-target illumination is used. Since the laser light in FIG. 9 isalso positioned normal to the target (as is the gas conduit) theobjective lens and the optical window have an opening for the gasconduit. After passing through the objective lens the conduit is bent totake the sample away from the optical path and move it into the ICP ionsource.

FIG. 10 shows an arrangement in which laser ablation and plume samplingis similar to the embodiment shown in FIG. 9. However, to avoid bendingthe gas conduit further downstream the laser light is bent instead usinga mirror. Here the optical window, the objective length and the mirrorall have openings for the passing of gas conduit carrying capture gasand plume material.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art. For example, in the variousexamples illustrated in the figures, the injector tube has beengenerally described with a 1 mm inner diameter in conjunction with thecumulative gas flow rate of about 1 liter per minute (0.1 plus 0.9 literper minute). It would be expected that smaller or larger diameterinjectors, along with the correspondingly selected gas flow rates, canbe applied to the various geometries presented with similar expectedresults. However, conditions for maintaining non-turbulent or nearlynon-turbulent gas dynamic within the injector tube in order forpreserving the distinctiveness of each separate ablated plume may bedesirable.

Furthermore, in some instances of elevated laser pulse rates, more thanone ablated plume can be distinctly captured and transferred to the ICPwithin the cumulative transit time spread as discussed above. Forexample, at a repetition rate of 10 kHz a pulsed laser can generate twoablated plumes in 200 μs that can be subsequently transferred to the ICPfor ionization. The ions generated from the two discrete plumes can beanalyzed as a single discrete packet of ions by the mass analyzer.Consequently, while the laser remains at the same ablation spot or whilethe laser's rate of movement over a trace of continuous spots is lessthan the repetition rate, the ablated plumes, and the subsequent ions,can provide an accumulative mass analysis at the same ablation spot orprovide an average mass distribution along the trace respectively. Itshould be noted that laser repetition rate as high as several MHz can beemployed resulting in a signal that represents averaging of many laserpulses. The laser can also be fired in bursts to provide a gap in thedata flow between individual sampling locations (or pixels).

It will be understood that the methods and devices of the invention maybe used with any of a variety of types of samples, e.g., biologicalsamples. In one approach the sample is cellular material, such as atissue section, cell monolayer, cell preparation, or the like. A samplemay be a thinly sectioned biological tissue up to 100 micrometersthickness, a tissue sample in the order of millimeters thickness, or anun-sectioned tissue sample. In one example, thin tissue sections (suchas paraffin embedded sections) may be used. For illustration, sometissue sections have a thickness of 10 nanometers to −10 micrometers. Insome cases, the sample is a group of cells, or one or more selectedcells from a group of cells. See, e.g., Antonov, A. and Bandura, D.,2012, U.S. Pat. Pub. 2012/0061561, incorporated by reference herein.

In some embodiments, the biological material is tagged with elementaltags, for example as described in U.S. Pat. Pub. US2010/0144056,incorporated herein by reference. A biological sample containing cells,proteins, cellular materials, of interest can be labeled with one, orseveral different, metal conjugated antibodies.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by thoseskilled in the relevant arts, once they have been made familiar withthis disclosure, that various changes in form and detail can be madewithout departing from the true scope of the invention in the appendedclaims. The invention is therefore not to be limited to the exactcomponents or details of methodology or construction set forth above.Except to the extent necessary or inherent in the processes themselves,no particular order to steps or stages of methods or processes describedin this disclosure, including the Figures, is intended or implied. Inmany cases the order of process steps may be varied without changing thepurpose, effect, or import of the methods described. All publicationsand patent documents cited herein are incorporated herein by referenceas if each such publication or document was specifically andindividually indicated to be incorporated herein by reference. Citationof publications and patent documents (patents, published patentapplications, and unpublished patent applications) is not intended as anadmission that any such document is pertinent prior art, nor does itconstitute any admission as to the contents or date of the same.

What is claimed is:
 1. A method of laser ablation mass cytometryanalysis using a laser ablation mass cytometer, the method comprising:directing pulses of a laser beam to a plurality of sites of a sample forgenerating an ablated plume of sample for each of the pulses; capturingeach ablated plume; transferring each of the captured ablated plumesinto an inductively coupled plasma (ICP); and ionizing each of thecaptured and transferred ablated plumes in the ICP, thereby generatingions for mass cytometry analysis; wherein the laser ablation masscytometer comprises an injector adapted to transfer the ablated plumesto the ICP, the injector having an injector inlet positioned within alaser ablation source, the injector inlet being configured for capturingthe ablated plumes; wherein the injector inlet forms a sample cone,wherein a narrower portion of the sample cone is an aperture of theinjector inlet; wherein the sample cone is positioned adjacent an areawhere the ablated plume is generated; and wherein the method furthercomprises introducing a capture gas flow to bring the ablated plumesinto the sample cone of the injector and introducing a transfer gasflow, separate from the capture gas flow, into the injector fortransferring the ablated plumes from the sample cone toward the ICP. 2.The method of claim 1, wherein the laser ablation mass cytometer furthercomprises: the laser ablation source for generating ablated plumes fromthe sample; an ICP source for producing the ICP; and a gas inlet coupledto the injector inlet configured to pass a gas from the gas inlet to theinjector inlet for transferring the captured ablated plume into the ICP.3. The method of claim 1, wherein the laser beam passes through theaperture.
 4. The method of claim 1, wherein the laser beam is from afemtosecond laser.
 5. The method of claim 1, wherein a position of thesample is changed during analysis; and wherein, during analysis, thelaser beam remains stationary.
 6. The method of claim 1, wherein thelaser beam pulses produce 1 micron ablation spots or less.
 7. The methodof claim 1, wherein the injector further includes a sacrificial flowportion where part of a sheath flow surrounding plume material isdiscarded before the plume material is introduced into the ICP.
 8. Themethod of claim 1, wherein a circumference of an outer surface of thesample cone decreases toward the aperture.
 9. The method of claim 1,wherein the sample comprises cells.
 10. The method of claim 9, furthercomprising labeling the sample with at least one metal conjugatedantibody.
 11. A laser ablation system comprising: a laser configured toproduce laser illumination; a laser ablation cell for holding a sampleto be analyzed; and an injector for carrying an ablation plume to anICP, the injector comprising a sample cone with a narrower portion ofthe sample cone forming an injector opening, and wherein the injectorcomprises an inlet for introducing a transfer gas flow into theinjector, and an outlet for delivering an ablation plume to ICP-MS; andwherein the laser ablation cell comprises an inlet for introducing acapture gas flow, separate from the transfer gas flow, into the ablationcell.
 12. The system of claim 11, further comprising a transfer gassource for producing a transfer flow in the injector and a capture gassource for producing a capture gas flow in the ablation cell.
 13. Thesystem of claim 11, wherein the laser is a femtosecond pulsed laser. 14.The system of claim 11, wherein the laser is a solid state laser. 15.The system of claim 14, wherein the solid state laser is an Nd:YAGlaser.
 16. The system of claim 11, wherein the laser illumination is alaser beam pulse that produces an ablation spot of 1 um or less.
 17. Thesystem of claim 11, further comprising an inductively coupled plasma(ICP) torch coupled to the injector, and a mass analyzer configured toreceive ions from the ICP torch.
 18. The system of claim 17, wherein themass analyzer is a time of flight mass spectrometer.
 19. The system ofclaim 11, wherein the injector further includes a sacrificial flowportion where part of a sheath flow surrounding plume material isdiscarded before the plume material is introduced into the ICP.
 20. Thesystem of claim 11, wherein the laser, the laser ablation cell, and theinjector are configured so that the laser illumination originates on oneside of the sample and the injector opening is on an opposite side ofthe sample.